Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR.

The G-->A transition at nucleotide 21881 of the human catechol-O-methyltransferase (COMT) gene represents a functional genetic polymorphism (Val158Met), rendering an enzyme with reduced activity that has been associated with psychiatric disorders and estrogen-related cancers. A new method for the detection of this polymorphism is described, based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), with a single PCR to discriminate both alleles. Two primers amplify a common amplicon independently of the allele considered. At the same time, two primers are used, differing in the 3' base. In the Val/Val or Met/Met conditions, amplification occurs both in the general amplicon and in the specific allele; in the Val/Met condition three different amplicons are produced. Direct DNA sequencing of a COMT region containing the G/A polymorphism demonstrates the validity of this tetra-primer ARMS-PCR method. Reevaluation by PCR-RFLP revealed 100% accordance for genotype adscription. Subjects carrying the COMT(HH) genotype in a Spanish population comprised 28%, and the COMT(LL) homozygotes amounted to 21%. The described method provides a fast and reliable approach for determining COMT polymorphism that can be useful in large clinical studies using minimal quantity of DNA, avoiding the timely and costly use of restriction enzymes.